Deposited reagents for chemical processes

ABSTRACT

Provided is a solid support having dry deposited thereon a first solid layer comprising at least a first compound, the compound for use in a chemical process conducted in a first solution. The invention allows stable forms where the first compound is not stable either (i) for storage in the first solution or (ii) in solution with one or more other compounds of the first layer. Also provided is a tray or kit of wells adapted for conducting a chemical process, each well having a deposited thereon a first solid layer comprising one or more compounds for supporting a chemical process conducted in a first solution, wherein addition of the first liquid to each of the wells dissolves said one or more compounds. Fabrication methods, time-release compositions, and methods of conducting chemical processes are further provided.

This application claims the benefit under 35 U.S.C. §119(e)(1) ofprovisional patent application Ser. No. 60/054,070, filed Jul. 29, 1997.

The present invention relates to methods of reliably and reproduciblydepositing reagents for conducting a chemical process onto a solidsupport, where in certain embodiments the reagents can be arrayed in apatterned array on the solid support, and to solid supports therebyproduced. Further provided are controlled release packets, which can bearrayed on a solid support, for delaying or controlling the time afterexposure of the containers to a liquid that it takes for the contents ofthe packets to dissolve. The methods of the invention use electrostaticsand electrical fields to produce the supports and controlled releasepackets.

In conducting a variety of clinical, forensic, environmental, research,quality control and other assays or chemical processes, it is oftendesirable to conduct a variety of parallel reactions or processes, forexample to accommodate a number of experimental samples and toaccommodate control reactions. Each of these reactions or chemicalprocesses typically needs a setup of the same reagents. Those who haveworked in a clinical or other science laboratory will recognize that oneof the most labor-intensive chores involves setting up an assay. Thischore is also one of the prime suspects for a source of variability inan assay. Recognizing this, Eastman-Kodak developed clinical analyzersthat take setup reagents from films produced by emulsion technologysimilar to that used to manufacture photographic films. These analyzersare now marketed by Johnson & Johnson Clinical Diagnostics (Raritan,N.J. and Rochester, N.Y.) as the Vitros brand analyzers. Emulsiontechnology is complex in its execution, and cannot readily form filmswith reagents that are not sufficiently stable or soluble in the wetemulsions used to produce the films. Further, this technology is limitedto applying reagents to films and is not well suited to applyingreagents in a pattern at separate locations on a support.

The present invention provides solid supports on which reagents forchemical processes are applied with a high degree of accuracy andreproducibility using electrostatic or controlled field deposition.Those reagents that are unstable in a solution can be deposited (a) as adry powder, (b) by use of a limited exposure to a wet toner vehicle, or(c) by selection of a wet toner vehicle in which the reagents are morestable. In any of these cases, the reagents are stored in a non-liquid("dry") form layered on the solid support. These deposition processesallow two reagents which typically react or are otherwise incompatiblewith one another to be stored on the same support. For example, wherethe reagents do not have significant vapor pressures they can bedeposited in the same layer while avoiding prolonged exposure to areaction-promoting solution form. Alternatively, multiple layers whichcan include separating layers can be applied so as to minimize theexposure of the two reagents to one another.

Further provided are packets for reagents or other compounds, whichreagents or other compounds are coated or admixed by controlled releaselayers. In one use, these reagents or other compounds can be releasedfrom the packets and into a liquid after other compounds have beendissolved. Thus, for example, a second antibody and detection reagentscan be released only after time and reagents have been provided forsupporting a binding reaction with a first antibody. Or in anotherexample, reagents are delayed from dissolving into an assay untilsufficient time has passed to allow experimental or control samples tobe added to all of the reaction vessels.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a solid support having drydeposited thereon a first solid layer comprising at least a firstcompound, the compound for use in a chemical process conducted in afirst solution. The invention allows stable forms where the firstcompound is not stable either (i) for storage in the first solution or(ii) in solution with one or more other compounds of the first layer.

In a second embodiment, the invention provides a tray or kit of wellsadapted for conducting a chemical process, at least one well (andpreferably two or more or all) has deposited thereon a first solid layercomprising one or more compounds for supporting a chemical processconducted in a first solution, wherein addition of the first liquid toeach of the wells dissolves said one or more compounds.

The invention further provides a method of fabricating a solid supporthaving deposited thereon a first solid layer comprising at least a firstcompound, the compound for use in a chemical process conducted in afirst solution, comprising

creating an electromagnetic force for attracting particles having afirst charge to a surface of the solid support, and

contacting the surface with the charged particles which comprise thematerial of the first layer.

The method can comprise: (1) in a first process, creating theelectromagnetic force by directing ions of a second polarity oppositethe first to the surface to create charges of the second polarity at thesurface; or (2) in a second process, creating the electromagnetic forceby generating an electrical field at a surface of the solid support. Inthese methods, the amount of material deposited can be monitored forinstance by monitoring depositions onto a sensing electrode ormonitoring the optical density or fluorescence or the depositedmaterial, and when a target amount of deposition has occurred removingthe electric field or removing non-adherent charged particles.

In a further embodiment, the invention provides a solid support havingdeposited thereon a first compound and a time-release composition,wherein upon exposure of the solid support to a first liquid in whichthe first compound is soluble the dissolution of the first compound isdelayed by the presence of the time-release layer. A layer of materialadded over the time-release composition can include a second compoundthat is dissolved more quickly than the first compound.

In still another embodiment, the invention provides a method ofconducting a chemical process in wells of a tray, wherein one or more ofthe wells is designated to receive a sample which can be dissolved inthe first liquid, the method comprising

(i) providing the wells, which have deposited therein a time-releasecomposition that comprises delayed-release reaction reagents that aresoluble in the first liquid,

(ii) adding first liquid to all of the wells, and

(iii) adding, for example concurrently with step (ii) or thereafter,sample to the designated wells such that each designated well receivessample prior to a designated time period after addition of the firstliquid to the well,

wherein the time-release composition assures that the delayed-releasereaction reagents are substantially delayed from dissolving in the firstliquid until after the designated time period.

Alternatively, the method can comprise

(a) providing the wells, wherein the time-release composition comprisesreaction reagents that are soluble in the first liquid, and wherein thesurface is further coated with a layer comprising early-release reactionreagents that are soluble in the first liquid, and

(b) adding first liquid to all of the wells and adding to sample to thedesignated wells,

wherein the time-release composition assures conditions for a firstreaction process are first supported by a dissolution of theearly-release reaction reagents and subsequently a dissolution of thedelayed-release reaction reagents assures conditions for a secondreaction process.

In another embodiment, the invention provides a solid support comprisingon a surface hereof a non-overlapping pattern of first solid layers eachcomprising a first compound for use in a chemical process conducted in asolution or in vapor phase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays a floating electrode apparatus.

FIG. 2 shows a patterned deposition of material A and a material B.

FIGS. 3A and 3B show a tray of wells in which materials have beendeposited.

FIG. 4 shows a substrate with controlled release features.

DEFINITIONS

The following terms shall have, for the purposes of this application,the meaning set forth below. In particular, for the purpose ofinterpreting the claims, the term definitions shall control over anyassertion of a contrary meaning based on other text found herein:

Attached

By "attached," "attachment," "attaching" and related words, theApplicants refer to bonding or adsorption of a compound to a surface ofa solid support of sufficient strength so that a liquid-solid phasechemical process can be conducted at the surface with the premise thatcompound will remain bonded to the solid support, or at least thatsufficient amounts of the compound will remain bonded so as not toundermine the intent of the process. For example, a chemical process maybe premised on the surface-bonded compound not being extracted into acontacting liquid, since for example the surface-bonded compound wouldnot be favorably present during later liquid-phase steps of the process;however, the degree to which extraction into the contacting liquid isdetrimental will depend on the particular process. Similarly, a chemicalprocess may be premised on having sufficient amounts of thesurface-bonded compound remaining available to play a role in generatinga surface-associated detection signal. In a preferred embodiment, atleast about 10% of the of the compound remains bonded to the surfaceafter the chemical process, more preferably at least about 20% remainsbonded, still more preferably at least about 50% remains bonded, yetstill more preferably at least about 80% remains bonded, and still yetmore preferably at least about 95% remains bonded. In a particularlypreferred embodiment, in excess of about 99% of the surface-bondedcompound remains bonded after the chemical process.

Dry Deposited

A material is "dry deposited" if deposited without applying the materialin a liquid vehicle.

Nomenclature for Covalently Attached Compounds

Where a compound is to be attached to a solid support by a covalentbond, this bond necessarily implies that the compound which is initiallydeposited and that which is eventually attached to the support are not,in a strict chemical sense, the same. However, for the purposes of thisapplication the deposited compound and the derivative formed incovalently attaching to the solid support are sufficiently the same,particularly where the property of the compound of interest ismaintained in the support-attached form.

Nucleic Acid

The nucleic acid sequences used in the invention are preferablydeoxyribonucleic acid sequences. However, they can also be ribonucleicacid sequences, or nucleic acid analogs, meaning compounds designed topreserve the hydrogen bonding and base-pairing properties of nucleicacid, but which differ from natural nucleic acid in, for example,susceptibility to nucleases.

Substantially Delayed

"Substantially delayed" from dissolving in the second liquid meansdelayed sufficiently so that, so long as the sample is added to a givenwell (reaction vessel) prior to a designated time period, atime-sensitive assay can be conducted based on the time that the firstliquid was added to the well rather than the time at which the samplewas added.

DETAILED DESCRIPTION OF THE INVENTION

Chemical, biochemical and molecular biological reactions require theaddition of many reagent components. These reagents can include enzymes,buffering agents, salts, organic and inorganic compounds andmacromolecules. Formulation of an optimized mixture of such reagents canbe challenging when the reagents are presented in liquid form. Manyreagents are not compatible at the required concentrations. Moreover,stability of the mixture as well as storage requirements imposeadditional challenges.

Currently, assay users are required to aliquot reagents from stocksolutions of reagents into a number assay vials. This process islaborious or requires expensive automation equipment, and is subject toerror.

1. Electrostatic and Controlled Field Deposition

In electrostatic deposition methods a substrate is sufficientlyelectrically isolated so that an electrostatic charge can be accumulatedon the substrate. One means of accumulating the charge is by takingadvantage of the photoelectric effect. In this method the substrate isexposed to electromagnetic radiation effective to strip charges,typically electrons, from the surface of the substrate. Other methodsinclude tribocharging, plasma treatment, induction charging and coronacharging. In a more preferred method, an ion emitter is oriented towardsthe surface on which one intends to create a charge and operated. Suchmethods of ion printing to controllably electrostatically depositcharged materials such as powders are described in detail in U.S.application Ser. Nos. 08/471,889 (filed Jun. 6, 1995), 08/659,501 (filedJun. 6, 1996) and 08/733,525 (filed Oct. 18, 1996), which documents areincorporated by reference herein in their entirety.

It should be noted that where the average charge-to-mass ratio of thecharged particles of the deposition material is known, the mass ofparticles that will effectively deposit can be relatively accuratelypredicted from the amount of charge previously accumulated on thesubstrate. In particular, for a given type of substrate a calibrationdatabase can be compiled. For a given average charge-to-mass ratio ofthe applied particles, the relationship of accumulated charge todeposited mass can be calibrated for a given set of materials andcharging conditions. In a production protocol, the averagecharge-to-mass ratio of the particles can be monitored, for instanceusing the velocimeter and a modified quartz crystal monitor described inU.S. application Ser. Nos. 08/661,211 and 08/661,210, both filed Jun.10, 1996, which documents are incorporated herein by reference in theirentirety. The illustrative charge-to-mass monitor functions by applyinga voltage to a crystal such as a quartz crystal to establish a vibratoryfrequency, monitoring changes in the vibratory frequency when exposed tothe charged particles, and correlating these changes to the mass of theparticles that impact the monitor. Another charge-to-mass monitor usesthe cage blowoff method of C. B. Schein and J. Cranch, J. Applied Phys.46: 5140, 1975. With the use of one or more charge-to-mass monitors,feedback loops can be incorporated into the electrical controls of adeposition apparatus. In one preferred embodiment, a charge-to-massmonitor is positioned so as to sample the charge-to-mass of particles attheir source (examples for source devices described below) and a chargemonitor (for example a device for measuring currents created by thedeposition of charged particles) is positioned adjacent to the site ofdeposition. The sampling values produced at these two sites providediagnostic data on the operation of the deposition apparatus.

A number of additional methods can be used to monitor the amount ofmaterial that is deposited on a solid support. For example, opticalmethods can include measuring reflectance, transmission, or fluorescenceusing laser or non-collimated light of broad or narrow band width. Othersources of directed electromagnetic energy can be used, for instanceX-ray absorption or fluorescence or microwave absorption can be used. Atuned circuit can be used to monitor an endpoint at which depositedmaterial creates a resonance with an energy source such as a microwaveenergy source. Acoustic absorption can also be used, where preferablythe sound source is an ultrasound source. Another exemplary measuringmethod can use a profilameter, which is a laser device that measures theamount the a beam of light is deflected by a surface with depositedmaterial to measure the depth of the deposited material. Furtherelectrical methods can include measuring a capacitance between aconductive material associated with the solid support (for example aconductive material incorporated into the solid support or a conductivematerial that has the solid support positioned adjacent to it) andanother conductor, where the deposited material is located between thetwo conductors.

A variety of additional factors can be monitored or controlled toincrease the reproducibility of the charge-to-mass ratios generated bythe charged deposition material source. For example, controlling thehumidity of the local environment, the nature and content of boundsolvent in the materials sought to be deposited, the purity of materialssought to be deposited, and the rubbing velocity effected in thetribocharging process can be important.

Another method of attracting charged deposition materials to a surfacehas been termed "controlled field deposition," and typically involvesapplying a potential to an electrode which directly or indirectlyresults in the formation of an attractive electrical field at thesurface upon which charged material will be deposited. For example, asubstrate can have electrical conductors positioned below the depositionsurfaces, and a potential applied to the conductors results in theformation of an attractive field at the surface. Where the separationbetween the substrate's surface and the conductors is sufficientlysmall, once an external potential is no longer applied to the conductorsthe charge of the deposition material results in a charge redistributionin the conductors such that an electrostatic "image" force is formedbetween the deposition material and the conductors, thereby helping tostabilize the deposition material's adherence to the surface.

Further examples of field-generating means include the use of "floatingelectrodes." A floating electrode is an electrode which develops alocalized field as a result of charge redistributions in the floatingelectrode, which are for example generated by voltages applied acrossone or more adjacent bias electrodes. Thus, for example, as illustratedin FIG. 1, a floating electrode apparatus 10 can have a backingelectrode 20, a non-conductive layer 30, a shielding electrode 60 and afloating electrode 70. In the illustrative floating electrode, a biaspotential applied across the backing electrode and the shieldingelectrode (which two electrodes serve as the bias electrodes) causes acharge redistribution in the floating electrode to created thecharged-particle attracting field at the floating electrode. Furtherdescription of floating electrodes and other forms of field generatingdevices for controlled field deposition can be found in U.S. applicationSer. No. 08/661,210, filed Jun. 10, 1996, which documents isincorporated herein by reference in its entirety. An advantage offloating electrode devices is that the amount of charged particles thatwill effectively adhere as a result of the field generated at thefloating electrode depends on the size of the bias potential. (For moredirect field generating apparatuses, the deposition can in principlecontinue for as long as a potential is applied.)

The field generating devices for controlled field deposition can bedesigned (a) to directly apply deposition material onto apparatuses thatincorporate electrodes for generating the field or (b) for use withelectrostatic chucks (i.e., field application structures) which operatein conjunction with the substrate on which deposition material is to beapplied. In the former case (a), it is generally desirable that themetallization processes used to create the electrodes is susceptible tomass production techniques. For example, the metallization can becreated by lithographic techniques where finely patterned electrodes aresought or by adhering or fusing metal layers to the substrate. In design(b), the electrostatic chuck is generally effective to electrostaticallyadhere the substrate to the chuck. This adherence of the substrate tothe chuck does not depend on the application of any process for creatinga charge on the substrate, but instead is believed to be the result of aredistribution of charges in the substrate in response to the fieldgenerated by the electrostatic chuck. Of course, a charge on thesubstrate can usefully be employed to strengthen electrostaticadherence. A third option is that the substrate is designed toreversibly couple with a device that provides the electrodes, such thatthe substrate and the coupled device provide a field-generatingapparatus. In this way, the electrode structures that can be a source ofmanufacturing costs remain separate from the consumable on whichreagents for conducting a chemical process will be deposited. Inaddition to the documents recited above, further information onelectrode structures and electrostatic chucks can be found in U.S.application Ser. No. 08/630,012, filed Apr. 9, 1996, which document isincorporated herein by reference in its entirety.

The charge of the particles applied to a substrate can be generated forexample by plasma treatment, radiation treatment (including treatmentwith suitably high energy electromagnetic radiation) or ion bombardment.More preferably, however, the charge is generated by tribocharging,wherein two materials with differing triboelectric constants rub againsteach other and transfer charge between one another. Tribocharging ismore preferred over the enumerated charge-producing methods because itexposes the particles to the least amount of reaction-promoting energy,and hence the tribocharging method is less susceptible to causingcompounds to degrade. Examples of materials that can be used fortribocharging include polytetrafluoroethylene ("TEFLON"), and polymersof chlorotrifluorethylene, chlorinated propylene, vinyl chloride,chlorinated ether, 4-chlorostyrene, 4-chloro-4-methoxystyrene, sulfone,epichlorhydrin, styrene, ethylene, carbonate, ethylene vinyl acetate,methyl methacrylate, vinyl acetate, vinyl butyral, 2-vinyl pyridinestyrene, nylon and ethylene oxide. See, for example,"Triboelectrification of Polymers" in K. C. Frisch and A. Patsis,Electrical Properties of Polymers (Technomic Publications, Westport,Conn.), which article is hereby incorporated by reference in itsentirety. For example, polytetrafluoroethylene and polyethylene andother negatively charged materials will generally create a positivecharge on an object. Nylon and other positively charged materials willgenerally create a negative charge on an object. Tribocharging andappliances for dispensing charged particles are describe in U.S.application Ser. Nos. 08/659,501 (filed Jun. 6, 1996) and 08/661,211(filed Jun. 10, 1996). U.S. application Ser. No. 08/661,211 describes,in particular, an acoustic dispenser that uses vibratory energy andgating electric fields to dispense charged particles for deposition ontothe substrate, and is incorporated herein by reference in its entirety.

In some embodiments, the charged particles may be made up of a wet tonerwherein particles of liquid material or liquid material with suspendedsolids are charged. Charging of the liquid particles can be by, forexample, tribocharging occurring at the time the particles are formed,utilizing contact potential differences between solid particles and theparticles, or modifying the differences in electrical potential usingsurface treatments such as surfactants. (See, L. B. Schein,Electrophotography and Development Physics, Laplacian Press, 1996, p.227.) Often it is favorable to dry deposit materials to avoid issues ofsolubility and stability of a chemical. On the other hand, however,liquid phase depositions are often practical, especially wherecautionary procedures, such as limiting the time of exposure to theliquid phase and selecting appropriate carrier solvents, are employed.Liquid phase distribution is for example useful where a material to bedeposited is not readily converted to a dry form that can be deposited,or where the non-deposited dry form does not retain an activity such asa biological activity.

2. Patterned Depositions and Removal of Excess Particles

Electrostatic or controlled field deposition methods can be used toapply patterns of materials on a substrate. For example, a pattern of andeposited material A and a deposited material B can be formed on asubstrate 100 as illustrated in FIG. 2. In some embodiments of theinvention, the deposition pattern can be highly dense, such as threehundred, six hundred or more dots per square inch (dpi). In preferredembodiments, the separation between the depositions is at least about 5μm and the width of the depositions is at least about 10 μm.

After the deposition process, it is in some embodiments desirable toremove nonadherent particles. This removal process can be particularlyimportant in embodiments where two separate patterns of depositionmaterial are applied to a substrate, since remnants of a material Acould possibly be found at the locations where a subsequent depositionof material B is anticipated. Methods to remove such nonadherent"background" particles can include rinsing (such as gentle rinsing witha sufficiently nonconductive and non-solubilizing solvent), blowing(such as gentle blowing with an inert gas), shaking, or application ofan electronic brush. An electronic brush is any device that is or can becalibrated and positioned to apply an electronic field that applies aforce on particles, where the field and resulting force can bemanipulated mechanically or electrically to displace nonadherent chargedparticles.

Referring again to FIG. 2, suppose for example that the substrate 100was conditioned to have a negative charge at the "A" sites by ionprinting. After positively charged particles of A material are applied,those particles that are do not adhere are removed. Ion printing canthen be applied to condition the "B" sites and apply the appropriatecharged particles of B material. As discussed further below, additionallayers can be applied to the substrate which can contain inertsubstances (inert to the use to which the substrate will be put), andthese additional layers often can be applied without the need forpatterned deposition or can be applied with reduced need for precisemetering of the deposition amount. Accordingly, these layers often canbe applied by methods other than electrostatic or controlled fielddeposition. For example, after the A material is deposited, thesubstrate is coated with layer of material to form an isolating layer,and thereafter the top layer of isolating material is conditioned by ionprinting to receive the B material.

3. Avoiding Unacceptable Levels of Adsorption to the Substrate

Where depositions are made directly on a substrate material (which forexample is not soluble in a liquid to which the substrate will later beexposed), at least an amount of the deposited material can be expectedto be attached to the substrate material. This effect will very with thedegree to which the substrate material tends to attach to substancesfound in the deposited material. In many instances the amount ofattached material will be small compared with the amount of materialthat can later be dissolved during the course of a chemical process, andthe percentage amount attached will be sufficiently reproducible so thatthe practical effect on the subsequent chemical process is negligible.However, these adsorption effects can be further minimized by coatingthe substrate with a soluble material, and then applying the depositionmaterial over this initial coating.

In two other applications, filed concurrently with the parent hereof andconcurrently herewith, Applicants describe the use of electrostatic andcontrolled field deposition to create defined amounts of attachedmaterials. See, copending patent application Docket No. SAR 12487,entitled "Solid Support With Attached Compounds," Loewy et al., which isincorporated herein by reference in its entirety. In certainembodiments, it is desirable to have certain compounds attached to thesubstrate, and other compounds, which may be present in an overlaidcoating, applied in a form that can be solubilized. For example, eachwell in a reaction tray can have attached to its bottom surface amacromolecule involved in an assay (such as an antibody, other receptormolecule, or a nucleic acid probe). A cocktail of the reagents neededfor at least the first step of an assay involving the macromolecule canalso be applied to a surface of the well, so that the addition of asolubilizing liquid provides a substantial beginning for the assay.

4. Supports, Vessels and Well Trays

Supports can be solids having a degree of rigidity such as glass,porcelain, silicon, plastic, and the like. Support can also be flexiblematerials such as plastic or otherwise synthetic materials, materials ofnatural polymers or derivatives thereof (such as cellulose or silk), andthe like. In certain embodiments the support is a porous material whichcan be rigid or flexible, such as sintered glass, intermeshed fibersincluding woven fabrics, and the like. In some embodiments, the solidsupport is a bead or pellet, which can be porous. In one embodimentwhere the support is a porous material the material of the supportbetween depositions is fused. In this way, the substrate is porous atthe portions where depositions have been made, but non-porous atintervening locations. The substrate thus has defined channels forallowing fluid flow through the substrate.

The substrate on which reagents are deposited can form part of a vesselin which a chemical process is to be conducted. In particular, thesubstrate can be a tray of wells such as is formed by molding processesof plastic or is created by etching or laser drilling techniques in avariety of materials (as described, for example, in U.S. applicationSer. No. 08/630,018, filed Apr. 9, 1996, which document is incorporatedherein by reference in its entirety). Such vessels can have associatedconductive layers which can form the electrodes used in controlled fielddeposition (where the conductive layer can for example couple withelectrical leads for providing electrical potentials) or provide aconductive layer supporting an image force to help retain chargedparticles. For example, FIGS. 3A and 3B illustrate substrates 201 and211, which include wells 202 and wells 212, respectively. Deposited inthe wells 202 and 212, are deposits 204 and 214, respectively. In FIG.3B, the deposits 214 are found in indentations (not numbered) found atthe bottom of wells 212. Underneath the wells 202 and 212 are conductivelayers 203 and 213, respectively, which conductive layers can support animage force for retaining charged particles. The deposits 214 are madeup of two layers, as indicated by a difference in shading.

In one embodiment of the invention, the support reagents are added tothe site at which the chemical process will occur in the form of apellet or other carrier that is added at the site of the chemicalprocess. For example, a pellet is added to each of a number of vessels,and liquid and sample materials are added to initiate the reactionprocess. In this case, the initial substrate on which the reagents aredeposited is selected so that such pellets (or other carriers) can bebuilt therefrom after the deposition process. Thus, for example, theinitial substrate can be a tablet or a capsule (into which materials canbe deposited). Alternatively, the initial substrate can be a sheet ofmaterial that can be cut into pellets or other carriers.

5. Alternative Methods of Applying Coatings

Additional layers can be applied to the substrate without electrostaticor controlled field deposition techniques. For example, coatingmaterials, which can be dry or more preferably dissolved or suspended ina volatile carrier, are applied by spraying, brushing, dipping or thelike. For dry powder depositions it will often prove desirable tomechanically scrap the top of the applied material assure that a uniformthickness of material has been applied. The coating material may forexample contain a low melting point polymer such as a polyethyleneglycol which is fused with moderate heat to more strongly bond theapplied layer of coating material to the substrate. Alternatively,sheets of material are applied for example using an intermediateadhesive or, where the materials are suitable, fusion bonding. Fusionbonding techniques include heat fusion, ultrasonic fusion, laser fusion,pressure bonding, and the like.

In some embodiments, the additional layers dissolve in the liquid of theanticipated subsequent chemical process.

6. Controlled Release

As an aspect of the invention, a reagent can be deposited such that itsrelease does not occur until after a time delay or until after a changeof conditions, such as a pH change, has occurred. In one form of theinvention, the controlled release operates to delay the operative phaseof a chemical process until all of the sites at which the process is tobe conducted in parallel have been fully formulated. For example, liquidcan be added to all of the sites, and then at least a subset of sitesreceives material from unknown samples or control samples. In one case,the simple addition of the liquid initiates a window time during whichto add all of the unknown or control material, after which time windowvarious reagents that support the chemical process are released into theliquid. Alternatively, a simple triggering event like a change of pHcould begin the release of the process-supporting reagents. Also,multiple layers of materials can be used so that, for example, a firstdeposited layer provides reagents that support a first chemical process,and thereafter another deposited layer releases reagents that support asecond chemical process. Such layered release layers can provide fortwo, three, or more phases of a chemical process.

Substantial development has been made, particularly with reference topharmaceuticals, in the field of controlled release or sustained releasecompositions. These compositions tend to be made up of mixtures ofpolymers with varying swelling properties and various excipients. Someof these compositions are designed with a focus on minimizing swellingin an acidic environment such as that of the human stomach, whileallowing faster swelling in an alkali environment, such as that of thesmall intestines. Particularly for veterinary applications, the pHdependence of the swelling profile can be reversed to favor swelling,and thereby dissolution of the active components of the composition, inacidic environments.

Examples of controlled release technology can be found in: (1) U.S. Pat.No. 4,012,498, "Sustained Release Formulations," Kornblum et al.,Sandoz, Inc. (contains alkaloids incorporated into a basic pH affectedcontrolled release matrix selected from cellulose acetate phthalate,polyvinyl acetate phthalate and hydroxy propylmethyl cellulosephthalate); (2) U.S. Pat. No. 4,111,202, "Osmotic System for theControlled and Delivery of Agent Over Time," Feliz, Alza Corp.; (3) U.S.Pat. No. 4,173,626, "Sustained Release Indomethacin," Dempski et al.,Merck & Co., Inc. (coats pellets with polyvinyl acetate to slowrelease); (4) U.S. Pat. No. 4,178,361 "Sustained Release PharmaceuticalComposition," Cohen et al., Union Corp. (uses a water-soluble but waterswellable matrix which holds a biological binding agent); (5) U.S. Pat.No. 4,221,778, "Prolonged Release Pharmaceutical Preparations,"Raghunathan, Pennwalt Corp. (ion exchange resin particles with drugabsorbed thereon which are treated with an impregnating agent[polyethylene glycol, propylene glycol, mannitol, lactose andmethylcellulose] to slow swelling in water and coated with a diffusionbarrier); (6) U.S. Pat. No. 4,248,857, "Sustained Release PharmaceuticalCompositions," DeNeale et al., American Home Products Corp.; (7) U.S.Pat. No. 4,252,786, "Controlled Release Tablet," Weiss et al., E. R.Squib & Sons, Inc. (medicament compressed with a blend of polymericvinyl pyrrolidone and a caroxyvinyl hydrophilic polymer and coated witha substantially water insoluble, but water permeable film); (8) U.S.Pat. No. 4,259,314, "Method and Composition for the Preparation ofControlled Long-Acting Pharmaceuticals," Lowey; (9) U.S. Pat. No.4,293,539, "Controlled Release Formulations and Method of Treatment,"Ludwig et al., Eli Lilly and Company (active dispersed in a copolymer ofglycolic acid and lactic acid); (10) U.S. Pat. No. 4,309,404, "SustainedRelease Pharmaceutical Compositions," DeNeale et al., American HomeProducts, Corp.; (11) U.S. Pat. No. 4,309,405, "Sustained ReleasePharmaceutical Compositions," Guley et al., American Home Products,Corp.; (12) U.S. Pat. No. 4,505,890, "Controlled Release Formulation andMethod," Jain et al., E. R. Squib & Sons, Inc. (a coated core containinga hydrocolloid gelling agent [methyl cellulose, hydroxypropyl cellulose,hydroxy ethyl cellulose, sodium carboxymethyl cellulose or mixturesthereof]; (13) U.S. Pat. No. 4,587,118, "Dry Sustained ReleaseTheophylline Oral Formulation," Hsiao, Key Pharmaceuticals, Inc., (seedcoated with theophylline and polyvinylpyrrolidone, then coated with amixture of ethylcellulose and hydroxypropylcellulose); (14) U.S. Pat.No. 4,666,705, "Controlled Release Formulation," DeCrosta et al., E. R.Squib & Sons, Inc.; (15) U.S. Pat. No. 4,716,041, "Diffusion CoatedMultiple-Units Dosage Form," Kjornaes et al., A/S Alfred Benzon(formulation is heated to form, in an film coating located inside anouter film layer, a continuous phase); (16) U.S. Pat. No. 4,784,858,"Controlled Release Tablet," Ventouras, Zyma SA (core contains watersoluble agent, a water-insoluble polymeric excipient [e.g.polyvinylchloride or polymer of lower alky acrylates or methacrylates],and a water-insoluble substance that swells on contacting water, andcore is coated with a elastic, water-insoluble, semipermeable diffusioncoating); (17) U.S. Pat. No. 4,917,900, "Controlled Release FormulationsContaining Zidovudine," Jones et al., Burroughs Wellcome Co. (coatedwith a mixture of a polymer of alkyl esters of acrylic or methacrylateand ethyl cellulose); (18) U.S. Pat. No. 4,973,469, "Drug DeliverySystem," Mulligan et al., Elan Corp., PLC (active ingredient and aninert substance whose aqueous solubility is inversely proportional tothat of the active are adsobed to a cross-linked polymer such ascross-linked polyvinylpyrrolidone, carboxymethylcellulose ormethylcellulose); (19) U.S. Pat. No. 5,178,868, "Dosage Form,"Malmqvist-Granlund et al., Kabi Pharmacia Aktiebolaq (cores coated witha mixture of (a) a copolymer of vinyl chloride/vinyl acetate/vinylalcohol monomers and (b), for creating pores, a substance that issoluble in water); (20) U.S. Pat. No. 5,234,691, "Sustained-ReleasePreparation of Basic Medicinal Agent Hydrochloride," Uemura et al.,Sumitomo Pharmaceuticals, Co., Ltd. (granules containing basic agent anda polyanion such a carboxyvinyl polymer or carboxymethcellulose andcoated with a slightly water-soluble macromolecular substance such aspolyvinyl acetate, ethyl cellulose, aminoalkylmethacrylate copolymer,methacrylic acid copolymer, cellulose acetates, polyethylene, polymethylmethacrylate, polydimethyl-siloxane, hardened oil, beeswax, carnaubawax, sucrose fatty acid ester, sorbitan monostearate, glycerylmonostearate, glyceryl monomyristate, glyceryl distearate, stearic acid,stearyl alcohol, and mixtures thereof); (21) U.S. Pat. No. 5,286,493,"Stabilized Controlled Release Formulations Having Acrylic PolymerCoating," Oshlack et al., Euroceltique, S.A. ((a) coating a substratewith a plasticized aqueous dispersion of ammonio methacrylate copolymerswhich are copolymerizates of acrylic and methacrylic esters, having alow content of quaternary ammonium groups acrylic and methacrylic acidesters, having a permeability which is unaffected by the pH conditionsprevailing in the gastrointestinal tract, and (b) curing the coatedsubstrate with a temperature greater than the glass transitiontemperature of the aqueous dispersion); (22) U.S. Pat. No. 5,472,712,"Controlled-Release Formulations Coated with Aqueous Dispersions ofEthylcellulose," Oshlack et al., Euroceltique, S.A.; (23) U.S. Pat. No.5,492,700, "Process and Composition for the Development of ControlledRelease Gemfibrozil Dosage Form," Ghebre-Sellassie et al.,Warner-Lambert Co. (a single granulation of gemfibrozil particlesgranulated with a release-control agent such as of cellulose phthalate,ethyl cellulose, polyvinyl phthalate, cellulose succinate, celluloseburyrate, poly(meth)acrylic acid, partially esterified poly(meth)acrylicacid and mixtures thereof); (24) U.S. Pat. No. 5,580,578, "ControlledRelease Formulations Coated with Aqueous Dispersions of AcrylicPolymers" Oshlack et al., Euroceltique, S. A.; (25) U.S. Pat. No.5,643,602, "Oral Composition for the Treatment of Inflammatory Bowel,"Ulmius, Astra Aktiebolag (a seed with a first coating of film-forming,water-soluble or insoluble polymers and a second coating of a membranecontaining a pharmaceutically acceptable, film-forming, anioniccarboxylic polymer which is difficult to dissolve at a low pH but issoluble at a higher pH of about 4 to 7.5); (26) U.S. Pat. No. 5,656,295,"Controlled Release Oxycodone Compositions," Oshlack et al.,Euroceltique, S.A, and (27) Ishikawa et al., Chem. Pharm. Bull. 43:2215-20, 1995 (describing polybenzylmethacrylate copolymer having across-linkable part on the side chain for use as an outer layer in acontrolled-release formulation, which copolymer is crosslinked forexample by contacting an oxygen plasma).

One focus of controlled release technology is in coating or mixingcompounds of interest with compositions that swell a given type ofliquid at a predictable rate. This technology relies substantially onthe swelling properties of polymers. Where one seeks to reduce theswelling rate in acidic aqueous environments, often the polymers usedinclude acid functional groups that titrate between a low solubilityacid form and a higher solubility salt form. Where one seeks to reducethe swelling rate in basic aqueous environments, often the polymers usedinclude base functional groups that titrate between a low solubilitybase form and a higher solubility salt form. It should be noted that theexcipients or fillers can play a role in modulating the rate at whichthe controlled release composition swells.

Additionally, the components of a controlled release formulation whichwill have an active role in a subsequent chemical process can affect thedissolution profile, as will be recognized by those of ordinary skill.The effects of these "actives" on the swelling profile can generally beexpected to be greater if admixed with the controlled releasecomposition rather than deposited under a layer of controlled releasecomposition.

The pH sensitivity of certain controlled release compositions can beutilized in designing protocols for chemical processes. For example, ifa first process is to occur at a low pH and a subsequent process at ahigher pH, the reagents that support the second process can besequestered by a controlled release composition that is more resistantto swelling in an acidic environment.

Another mechanism for controlling release is to provide akinetic/diffusion barrier to substrate deposited chemicals passing intoa liquid. For example, FIG. 4 illustrates a substrate 301 in whichmaterials have been deposited in cavities 302, which in turn is coveredby a membrane 303. The substrate is made up of a lower portion 305, towhich is fused an upper portion 306. The upper portion defines wellsabove the locations of the cavities. This diffusion control mechanismcan of course be combined with the rate-of-swelling mechanism discussedabove. As alluded to above in the recitation of published examples ofcontrolled release formulations, the diffusion control can be formed aspolymer-containing films overlying an interior composition.

7. Methods and Substrates for Handling Incompatible Reagents

In many cases reagents that are used together in a chemical process arenot stable if stored together, especially in solution. This lack ofstability (at least in the long term) is often attenuated when reagentsare stored in a solid form such that opportunities for the reagents tocollide are minimized. By the present invention, the reagents can bedeposited by a dry deposition method or, if liquids are used in thedeposition process, the time during which the reagents are solubilizedor suspended in the liquids can be kept to a minimum. Using theintermediary layers described above, and even intervening layers ofcontrolled release compositions, the incompatible reagents can befurther separated. By simply depositing the reagents in separatedeposition steps, the exposure of the reagents to one another is reducedeven where the incompatible reagents are deposited in adjacent layers.

Reagents can be incompatible in the sense that one is favorablyprocessed in a liquid in which a second reagent is insoluble orunstable. This contingency can be addressed by the present invention byhaving the reagents both applied by a dry deposition method, or byhaving the second reagent applied by a dry deposition method.

8. Preferred Chemical Processes

One example in which controlled release is used is in processes thatrequire several enzyme catalyzed reactions. The product of one enzymereaction serves as substrate for a different enzyme used in a secondprocess step. Each enzyme has differing requirements relating forexample to salts, buffers, cofactors, temperature and the like. Forexample, an nucleic acid amplification (such as a polymerase chainreaction) can be initiated by the enzyme reverse transcriptase, whichhas certain requirements of pH, salts, temperature and the like. Afterthe initial reverse transcriptase reaction, a DNA polymerase can beused, which polymerase enzyme has different requirements than thereverse transcriptase. Moreover, during the subsequent DNApolymerase-mediated operations it is desirable to assure that thereverse transcriptase is no longer functioning under sub-optimumconditions, so the controlled release formulation can be designed torelease reverse transcriptase inhibitors.

Nucleic acid amplification methods include without limitation (1)Polymerase chain reaction (PCR; see, e.g., U.S. Pat. No. 4,683,202 andShort Protocols In Molecular-Biology (Frederick M. Ausubel et al., eds.1992)(hereinafter, Ausubel et al.), Unit 15.1); (2) ligase chainreaction (LCR; see, e.g., European Patent Publication 320,308 andSchachter et al., J. Clin. Microbiol., 32, 2540-2543 (1994)); (3) stranddisplacement amplification (SDA; see, e.g., Walker et al., PCR Methodsand Applications, 3, 1-6 (1993)); (4) nucleic acid sequence-basedamplification (NASBA; see, e.g., van Gemen et al., J. Virol. Methods,43, 177-188 (1993)); and (5) transcription-mediated amplification (TMA;Pfyffer et al., J. Clin. Micro., 34, 834-841 (1996)). For example, therecan be three nucleic acid segments (the segment of double-stranded DNAto be amplified and two single-stranded oligonucleotide primers flankingthis segment), a porotein component (a DNA polymerase), appropriatedNTPs, a buffer and salts. The procedures for these amplificationmethods are described for example in the above-cited documents, and thisdescription of methodology is incorporated by reference in the presentdisclosure. Further description of amplification methodology is found inMyers and Sigua, "Amplification of RNA: High Temperature ReverseTranscription and DNA Amplification with Thermus Thermophilus DNA," inPCR Strategies, Academic Press, 1995, which document is incorporatedherein by reference in its entirety.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations in the preferred devices and methods may be used andthat it is intended that the invention may be practiced otherwise thanas specifically described herein. Accordingly, this invention includesall modifications encompassed within the spirit and scope of theinvention as defined by the claims that follow.

What is claimed:
 1. A solid support having dry deposited thereon two ormore separated regions of a first solid layer comprising at least anonvolatile compound, the nonvolatile compound for use in a chemicalprocess conducted in a first solution wherein the first layers aredeposited by electrostatic or controlled field deposition.
 2. The solidsupport of claim 1, wherein the nonvolatile compound is a macromolecule.3. The solid support of claim 1, wherein the nonvolatile compound is notstable either (i) for storage in the first solution or (ii) in solutionwith one or more other compounds deposited on the solid support so as toparticipate with the nonvolatile compounds in the chemical process. 4.The solid support of claim 3, wherein the nonvolatile compound is amacromolecule.
 5. The solid support of claim 1, wherein each of two ormore separated regions of first solid layer comprises a distinctnon-volatile compound.
 6. The solid support of claim 5, wherein thenonvolatile compound is a macromolecule.
 7. A tray of wells adapted forconducting a chemical process, a plurality of the wells having drydeposited thereon a first solid layer comprising one or more nonvolatilecompounds for supporting a chemical process conducted in a firstsolution, wherein addition of the first liquid to each of the wellsdissolves said one or more nonvolatile compounds wherein the firstlayers are deposited by electrostatic or controlled field deposition. 8.The tray of wells of claim 7, wherein the first layers comprise, in anamount adapted for use in a nucleic acid amplification method (i) a DNApolymerase or a reverse transcriptase and (ii) a oligonucleotide primer.9. The tray of claim 7, wherein one or more of the nonvolatile compoundsis a macromolecule.
 10. A solid support comprising on a surface thereofa non-overlapping pattern of dry deposited first solid layers eachcomprising a first nonvolatile compound for use in a chemical processconducted in a solution phase wherein the first layers are deposited byelectrostatic or controlled field deposition.
 11. The solid support ofclaim 10, wherein the first nonvolatile compound is a macromolecule. 12.The solid support of claim 10, further comprising on the surface anon-overlapping pattern of dry deposited second layers each comprising asecond nonvolatile compound for use in a chemical process conducted in asolution.
 13. The solid support of claim 12, wherein the secondnonvolatile compound is a macromolecule.